Methods for isothermal molecular amplification with nanoparticle-based reactions

ABSTRACT

The present method of detection involves increasing an amount of analyte molecules by an isothermal molecular amplification approach. In the present approach a starting molecule of interest may be amplified through a reaction it induces with specifically engineered and functionalized particles, namely protected particles A and storage particles B. This reaction may result in a set of output DNA molecules that is larger in number than the input DNA molecules. Thus the reaction between nanoparticles for amplification of a certain DNA sequence (input DNA molecules) may occur when there is a match with a targeted molecule (stored molecules on storage particles B,) and if the DNA sequence of the input DNA molecules does not match (partially or completely) the targeted molecule the reaction may not occur. Without a certain molecular input of the input DNA molecule the reaction may not occur.

CROSS-RELATED APPLICATION

The present application claims priority from U.S. ProvisionalApplication No. 62/508,682, filed May 19, 2017, which is incorporatedherein by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

The present application was made with government support under contractnumber DE-SC0012704 awarded by the U.S. Department of Energy. The UnitedStates government has certain rights in the invention(s).

FIELD OF THE INVENTION

This application relates to isothermal molecular amplification withnanoparticle-based reactions.

BACKGROUND OF THE INVENTION

Bio-sensing, counterfeit detection and chemical safety require sensitivedetection of minute amounts of target molecules. That is, such detectionrequires complex and costly methods to reveal infinitesimal amounts ofmaterials. In some cases, single molecule level detection may beachieved through optical methods. However challenges may remain for useof such optical methods for practical real-life applications. Othermethods of detection may use physical detection such as, for example,fluorescent detection, Raman signal, plasmonic shift or the like.

State of the art physical detection methods focus on the detection sideof molecular sensing, i.e. they are based on enhancing the sensitivityof the detection apparatus or applying methods that permit moresensitive detection modes/principles. However, there is still a need toaddress the other side of the detection process by increasing theincoming molecular signal.

SUMMARY OF THE INVENTION

The present isothermal molecular amplification approach relates to amethod of detection by increasing an amount of analyte molecules. In thepresent approach a starting molecule of interest (e.g., DNA withspecific input sequence as the analyte molecule or referred to also asan input DNA molecule (yellow)) may be amplified through a reaction itinduces with specifically engineered and functionalized particles,namely protected particles A and storage particles B. This reaction mayresult in a set of output DNA molecules (yellow) that is larger innumber than the input DNA molecules (yellow).

The reaction between nanoparticles for amplification of a certain DNAsequence (input DNA molecules (yellow)) may occur when there is a matchwith a targeted molecule (stored molecules (yellow)) on storageparticles B, and if the DNA sequence of the input DNA molecules (yellow)does not match the targeted molecule the reaction may not occur. Withouta certain molecular input of the input DNA molecule (yellow) thereaction may not occur.

The amplification by this reaction may be by a factor of about 100-500,and larger factors may be achieved through optimization and systemdesign that one skilled in the art may carry out. The present isothermalmolecular amplification system may be utilized with known detectionmethods, thus, it may enhance the sensitivity of other methods.

A sensor apparatus for detecting targeted molecules comprising one ormore protected particles A, storage particles B and input DNA moleculeswherein protected particles A comprise first sequence (red) strands andsecond sequence (green) strands functionalized thereon, storageparticles B comprise third sequence (purple) strands grafted thereon andstored targeted DNA molecule (yellow) partially hybridized thereon,protected particles A, deprotected by input DNA molecules, react withstorage particles B by a duplex between third sequence (purple) strandsand first sequence (red strands), and wherein input DNA molecules have amatching DNA sequence with stored targeted DNA (yellow) molecules andoutput DNA molecules (yellow).

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and other features of this disclosure will become morefully apparent from the following description and appended claims, takenin conjunction with the accompanying drawings. Understanding that thesedrawings depict only several embodiments in accordance with thedisclosure and are, therefore, not to be considered limiting of itsscope, the disclosure will be described with additional specificity anddetail through use of the accompanying drawings, in which:

FIG. 1(a) shows an energy landscape of present isothermal DNAamplification and illustration of an example of an overall scheme of anisothermal molecular amplification reaction system;

FIG. 1(b) shows DNA replacement reaction induced aggregation andisothermal amplification of yellow DNA molecule (about 50 strands per 10nm particle);

FIG. 2(a) shows schematic representation of origami protected particleA;

FIG. 2(b) AFM topography of the construct respectively. The scale bar is200 nm;

FIG. 2(c) shows a histogram of the number of origami attached to the 20nm AuNP A (bar) and a Gaussian function fitting (line curve) to thenumber distribution, on average there are approximately 3-4 origamiattached to each DNA coated AuNP A;

FIG. 3(a) is a schematic representation of origami protected particle A;

FIG. 3(b) is a schematic representation of origami protected particle Aand storage particle B;

FIG. 3(c) is a schematic representation of origami protected particle A,storage particle B and input DNA molecule (yellow strand);

FIG. 3(d) DLS hydrodynamic diameter of construct of system shown inFIGS. 3(a)-(c): origami protected particles A (FIG. 3(a)) shown by curvelabeled (a); origami protected particles A and storage particles B (FIG.3(b)) mixed but not activated by input DNA molecule (yellow strand)shown by curve labeled (b); origami protected particles A, storageparticles B with added input DNA molecule (yellow strand) (FIG. 3(c))added in different concentrations—10 nM shown by curve labeled (c1), 100nM shown by curve labeled (c2), and 1000 nM shown by curve labeled (c3)respectively;

FIG. 4(a) shows a schematic representation and a change of fluorescenceof storage particle B (2.1 nM) with the addition of excess of firstsequence (red) DNA strand;

FIG. 4(b) shows a schematic representation and a change of fluorescencewith a 1:1 ratio of protected particles A and storage particles B (2.1nM each), and 0.5 nM of input DNA molecules (yellow strands) combinedtogether;

FIG. 4(c) relative change of fluorescence intensity with increasingamount of first sequence (red) DNA strand added shown as black squares.The x-axis intercept line is a straight black dotted line labeled as thenumber of yellow DNA grafted/particle and corresponds to the number ofstored input molecules (yellow DNA strands) on storage particles B. Thechange in the intensity for the fluorescence enhancement assayexperiments for origami protected particles A and storage particlesB-Y-Cy5 is approximately 1.2 (portion of dotted line parallel to x axisand labeled “Number of yellow DNA released/particle”) corresponds to40-50 output DNA molecules (yellow strands) released per particle(portion of dotted line parallel toy axis and labeled “Number of yellowDNA released/particle”);

FIG. 4(d) shows the kinetics of the present amplification reaction,monitored by in situ fluorescence and DLS measurements; the reaction iscompleted within 1 hour of initiation;

FIG. 5 shows the effect of DNA mutation on the aggregation. A+Brepresents a 1:1 ratio of origami protected particles A and storageparticles B-Y-Cy5. A+B+Y represents when 10 nM of input DNA molecules(yellow) are added to a solution of A+B leading to an aggregation of Aand B particles. Presence of 10 nM of input DNA molecules (yellow) withthree mutations (A+B+Y3), two mutations (A+B+Y2) and one mutation(A+B+Y) failed to produce any aggregations; and all arranged accordingto at least some embodiments described herein.

DETAILED DESCRIPTION OF THE INVENTION

In the following Detailed Description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented herein. It will be readily understood that the aspects of thepresent disclosure, as generally described herein, and illustrated inthe Figures, can be arranged, substituted, combined, separated, anddesigned in a wide variety of different configurations, all of which areexplicitly contemplated herein.

It will be understood that any compound, material or substance which isexpressly or implicitly disclosed in the specification and/or recited ina claim as belonging to a group or structurally, compositionally and/orfunctionally related compounds, materials or substances, includesindividual representatives of the group and all combinations thereof.

The present oligonucleotides can be any type of oligonucleotide, eithernaturally-occurring or artificial. Typically, the oligonucleotide is DNAor RNA.

In one embodiment, an isothermal molecular amplification approach isprovided by which a molecule of interest (e.g., DNA with a specificsequence as an analyte molecule, or referred to also as an inputoligonucleotide molecule (yellow) or “Input-Oligonucleotide”) may beamplified to produce output molecules (i.e.,“Stored-Input-Oligonucleotide”) with the same or differentoligonucleotide (e.g., DNA) sequence through a reaction that may beinduced between specifically designed and oligonucleotide functionalizedparticles, namely protected particles A (i.e., “Particle-A”) and storageparticles B (i.e., “Particle-B”). As a result, more outputoligonucleotide molecules (yellow), of the same sequence as the inputmolecules (yellow), may be produced than the input oligonucleotidemolecules (yellow) originally added. The molecule of interest may be atargeted or analyte input DNA molecule and may be designated as yellowstrands. The input molecule may be a single stranded oligonucleotide(e.g., DNA) sequence, e.g., DNA with a length of between about 10 to 120bases. Input DNA molecules (yellow) may be added to initial or partiallydeprotected particles A. A mixture of protected particles A comprisingoligonucleotide origami and storage particles B can be prepared,formulated and/or provided.

Initial or Deprotected Particles A (I.e., Deprotected “Particle-A”)

Initial or deprotected nanoparticles or particles A may befunctionalized with first sequence oligonucleotide (e.g., DNA) strandsand second sequence oligonucleotide (e.g., DNA) strands. First sequenceand second sequence strands may be complementary in sequence to oneanother. The first sequence strand may be designated as red strands (or“Oligonucleotide-1”) and the second sequence strand may be designated asgreen strands (or “Oligonucleotide-2”). Thus, second sequence strand(green strands) may be complementary in sequence with first sequencestrands (red strands.) First sequence strands and second sequencestrands (i.e., green and red strands) may partially hybridize to form“Duplex 1-2”. Typically, Oligonucleotide-1 is grafted to Particle-A.

Partially hybridized first sequence strands and second sequence strandsgreen and red strands) may facilitate binding of oligonucleotide (e.g.,DNA) origami to initial or deprotected particles A. Oligonucleotideorigami may protect initial or deprotected particles A from reacting toform aggregates or binding with targeted particles B. Whenoligonucleotide origami binds to initial or deprotected particles A theyare transformed to protected particles A.

Protected Particles A (I.e., Protected “Particle-A”)

The design of protected nanoparticles or particles A may render themnonreactive and protected from aggregation with other particles, namelystorage particles B (i.e., “Particle-B”). The design of protectedparticles A may be rendered reactive with the addition of an inputoligonucleotide molecule (yellow). The protected particle A when reactedwith the input oligonucleotide molecule (yellow) (i.e.,“Oligonucleotide-Y”) may become partially or fully deprotected andamplification may be enabled. The amplification reaction may proceeduntil the deprotected particles A are consumed/reacted and no morerelease is possible (i.e., substantially all or all stored inputoligonucleotide molecules are released and the reaction ends). Fullydeprotected particles A cannot bind to input oligonucleotide molecule(yellow).

The protected particles A may have a shell or a shield ofoligonucleotide origami. Oligonucleotide origami may act as a protectiveshield (“umbrella” or “blocking plates”) for initial or deprotecteddesigned particles A transforming them to protected particles A.Blocking plates may comprise oligonucleotide (e.g., DNA) origami.Oligonucleotide origami may be bound to initial or deprotected designedparticles A. In some embodiments, oligonucleotide origami may be planarand have a shape that is rectangular, round or other designed2-dimensional constructs, or may be a 3-dimensional construct, such as,for example, polyhedron shaped. Planar oligonucleotide origami platesmay be bound to initial designed nanoparticles or deprotected particlesA may act as a protective shield/shell (“umbrella” or “blockingplates”). The oligonucleotide origami may be a coating on particles A.

Oligonucleotide origami may provide steric shielding againsthybridization between oligonucleotide strands (e.g., first sequence(red) strands, i.e., “Oligonucleotide-1”) bound to particles A andoligonucleotide strands bound to other particles (e.g., storageparticles B). Specifically, oligonucleotide origami may provide stericshielding against hybridization of third sequence (purple or“Oligonucleotide-3”) strands on storage particles B and first sequence(red or “Oligonucleotide-1”) strands on initial or deprotected particlesA. Such a design of the shell may prevent aggregation of particles Awith, for example, storage particles B in the solution when no input DNAmolecule (yellow or “Input-Oligonucleotide”) is present. Theoligonucleotide origami shape can be designed by one skilled in the artto maximize the particle A shielding or optimize other reactionparameters (e.g. DNA hybridization with input DNA).

Storage Particles B (I.e., “Particle-B”)

Storage nanoparticles or particles B (i.e., Particle-B) may befunctionalized with third sequence strands. The third sequence strandsmay be grafted onto storage particles B. The third sequence strands maybe designated as purple strands (or “Oligonucleotide-3”). Third sequence(purple) strands may form a duplex with first sequence (red) strands ofinitial or deprotected particles A, i.e., when particles A are notprotected by oligonucleotide origami. Such duplex is referred to as“Duplex 1-3.” Storage particles B may also have stored input DNAmolecules, designated as yellow strands hybridized partially on them,e.g., a hybridization between “Oligonucleotide-3” and“Stored-Input-Oligonucleotide” to form “Duplex 3-Y.” Stored inputoligonucleotide molecules (yellow) partially hybridized on storageparticles B may have the same or different oligonucleotide sequence asinput oligonucleotide molecule (yellow). If it is a different sequence,then there may be a portion or part that is different and a portion orpart that is the same. For example, less than about 50%, about 40%,about 30%, about 20, about 10% or about 5% can be different.

Amplification Reaction

The reaction between specifically engineered andoligonucleotide-functionalized nanoparticles, namely partiallydeprotected particles A (i.e., “Particle-A”) and storage particles B(i.e., “Particle-B”), may result in amplification of a certainoligonucleotide (e.g., DNA) sequence of stored input DNA molecules(yellow) on storage particles B if they match a targeted or analyteinput DNA molecule (yellow) and a cascade reaction is induced. On theother hand, if the oligonucleotide sequence of stored input DNAmolecules on storage particles B do not match the targeted inputoligonucleotide molecule, no reaction may take place. No reaction mayoccur without addition of a targeted or analyte input DNA molecule topartially deprotected particles A and storage particles B.

An input oligonucleotide molecule (yellow) (e.g., a single stranded DNAsequence, “Input-Oligonucleotide”) may induce an amplification reactionbetween certain designed initial or partially deprotected particles A(“Particle-A”) and storage particles B (“Particle-B”). The reaction mayproduce and result in a release of new output DNA molecules (yellow)stored on storage particles B (“Stored-Input-Oligonucleotide”). Wheninput oligonucleotide molecules (yellow) are added to protectedparticles A, the protected particles A may be deprotected. Whenprotected particles A are partially deprotected, an amplification orcascade reaction may produce output oligonucleotide molecules (yellow)in a much larger number/amount than the input oligonucleotide molecules(yellow) that were added to start the reaction. In turn, the releasednew output DNA molecules may act as input DNA molecule and promote oneor more follow-on (second, third, and so on) reaction(s) and newadditional output DNA molecules (species) of the same sequence (T1) ordifferent sequence (T2) may be produced and released. The cascadereaction may repeat until protected particles A are fully deprotected.

As used herein amplification refers to a number of outputoligonucleotide (e.g., DNA) molecules that is larger than inputoligonucleotide molecules. The amplification by the present reaction maybe by a factor of 100-200, 100-300, 100-400, or 100-500 and largerfactors depending on the details of the desired nanoparticle design,i.e. it size, sequence length, environmental parameters etc. Theamplification factor (i.e., ratio of output number of molecules to inputnumber of molecules) may depend on the size of nanoparticles A and B(about 10-20 nm), design of the shell and protection shield (DNAorigami), and reaction parameters (e.g., salt, particle concentration,DNA, temperature). The amplification reaction may occur withouttemperature ramping. The operational temperature for the “amplifier” maybe determined by nanoparticle design, as would be known to a skilledartisan, so it may be referred to as an isothermal amplification. Theoperational temperature may be room temperature.

The input molecules (yellow) are capable of deprotecting a subset of theprotected particles A partially by removing one or more ‘umbrella’oligonucleotide (e.g., DNA) origamis (“blocking plates”). The protectedparticles A may be deprotected by release of blocking plates. Afterparticles A are deprotected, a duplex between third sequence (purple)strands on particles B and first sequence (red) strands on particles Amay be energetically favorable (i.e., “Duplex 1-3”). Deprotectedparticles A and storage particles B may bind when first sequence (red)and third sequence (purple) strands are able to hybridize. Multiplebinding of particles A and B may result in particle aggregation ratherthan isothermal amplification.

For example, after adding 0.5 nM input DNA (yellow) molecules, thereaction between partially deprotected particles A and storage particlesB (having stored input (yellow) strands partially hybridized on it) maybe activated. Stored input DNA (yellow) strands on storage particles Bmay be hybridized with third sequence (purple) strands that are graftedon. The inter-particle reaction between partially deprotected particlesA and storage particles B may produce and release new output molecules(yellow) of the same or different type (that have been stored on theparticles B). In turn, the newly released output oligonucleotidemolecules (yellow) may further promote inter-particle reaction that maylead to further releases of output oligonucleotide molecules (yellow).Thus, amplification of the input DNA molecules (yellow) in the cascadeof “chain”-like reactions may be obtained. (The amount of input DNAmolecule (yellow) may be, for example, about 0.5 nM to about 1000 nM.)

As depicted in FIG. 1(a), the energy landscape of the present isothermaloligonucleotide molecule amplification system may relate to a transitionfrom a higher energy metastable state to a lesser energy configurationto a lowest energy state. This transformation through the energy statesfrom a high energy state to low energy state may be via a sequentialtoehold mediated strand displacement reaction by input oligonucleotidemolecule (yellow). The input oligonucleotide molecules (strands) may beutilized to overcome an initial energy barrier, or may be utilized toactivate the amplification reaction through for example, deprotection(i.e., release) of blocking plates, made out of oligonucleotide origami.

In one embodiment, input molecules and output molecules may be the sameand amplification of the same type (noted as ‘T1’) may occur. Theamplification reaction may be activated through deprotection (i.e.,release of blocking plates made out of oligonucleotide origami) ofparticles A and the reaction may feed itself: additional strands ofoutput molecules may be released while A and B particles begin toaggregate due to binding of oligonucleotide on their surfaces. Thereaction may end when substantially all or all initial particles A arereacted and output molecules (the same type as the input molecule, butlarger amount than the input molecules) are released.

In one embodiment, multiplication of other predefined molecules may beachieved, whereby an input molecule ‘T1’ may result in the release ofoutput molecules ‘T2’ (stored on storage particles B), and the amount of‘T2’ may be larger than ‘T1’. The amplification factor may be defined asa ratio of the number/concentration of output molecules to thenumber/concentration of input molecules, i.e. [T2]/[T1].

Another Embodiment

In one embodiment, a method for amplifying an Input-Oligonucleotide isprovided. The Input-Oligonucleotide can be of any length. Typically, theInput-Oligonucleotide is about 5 to about 200 bases, or about 10 toabout 120 bases. Typically, the Input-Oligonucleotide is an unknownsample of oligonucleotides that is to be identified.

The method includes the use of a plurality of particles. The particlescan be any type of particle to which oligonucleotides can be grafted.Typically, the particles can have diameters in the nanometer to micronrange. For example, nanoparticle can range from about 5 to about 100 nmin diameter. Typically, the particles are metallic or semiconducting.Examples of suitable particles include gold (Au), silver (Ag), copper(Cu), platinum (Pt), palladium (Pd) and combinations thereof. Aplurality of such particles is functionalized with oligonucleotides.Particles which are functionalized with the same oligonucleotides havethe same designation.

For example, a plurality of particles, designated as Particle-A, can befunctionalized with a plurality of Oligonucleotide-1 and a plurality ofOligonucleotide-2. Typically, Oligonucleotide-1 is grafted ontoParticle-A. There are about 25 to about 400 Oligonucleotide-1 attachedper about 5 nm to about 40 nm diameter of Particle-A. Typically, thereare about 200 Oligonucleotide-1 attached per about 20 nm diameter ofParticle-A. Oligonucleotide-1 is hybridized to Oligonucleotide-2 to formDuplex 1-2. Typically, Duplex 1-2 is formed by a partial hybridization.

Oligonucleotide origami is attached to Duplex 1-2 to render Particle-Anon-reactive (i.e., protected). The origami can be any size whichprotects Particle-A. The origami is 2-dimensional or 3-dimensional, forexample, the origami can be rectangular. A typical size of the origamiis about 5 to about 100 nm per about 20 nm of particle diameter, forexample, about 50 nm in length per about 20 nm diameter of Particle-A.Due to selective hybridization between Oligonucleotide-1 andOligonucleotide-2, Particle-A may bind to several origami structures.Typically, there can be about 2 to about 10 origami per about 5 nm toabout 40 nm diameter of Particle-A. For example, there can be about 3 toabout 4 origami per about 20 nm diameter of Particle-A.

Additionally, a plurality of particles, designated as Particle-B, can befunctionalized with a plurality of Oligonucleotide-3 and a plurality ofStored-Input-Oligonucleotide. Typically, Oligonucleotide-3 is graftedonto Particle-B. Typically, there are about 25 to about 400Oligonucleotide-3 attached per about 5 nm to about 40 nm diameter ofParticle-B. For example, there can be about 200 Oligonucleotide-3attached per about 20 nm diameter of Particle-B. Oligonucleotide-3 ishybridized to Stored-Input-Oligonucleotide to form Duplex 3-Y.Typically, Duplex 3-Y is formed by a partial hybridization. Typically,the hybridization does not take place at the ends of the oligonucleotidestrands; that is, hybridization takes places within the strands,referred to as “inter-strand” hybridization.

In one embodiment, at least a portion of the sequences ofInput-Oligonucleotide and the Stored-Input-Oligonucleotide areidentical. For example, the sequences are at least about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, about 97%, about 98%, about99% or 100% identical.

The Input-Oligonucleotide, Particle-A and Particle-B are mixed. Themixing can occur in any order. Typically, the reaction is underisothermal conditions. For example, the whole reaction can occur atabout room temperature. Typically, the relative amount ofParticle-A:Particle-B is about 10:1 to about 1:10, e.g., about 1:1.Typically, the relative amount of Input-Oligonucleotide to Particle-A isat least about 1:4. The amount of Input-Oligonucleotide to Particle-Acan be in vast excess, e.g., about 1000:4

Typically, the Input-Oligonucleotide is added to Particle-A first. Oncemixed, a cascade reaction occurs so that: i) the Input-Oligonucleotidehybridizes with Oligonucleotide-2 to form Duplex Y-2, thereby removingOligonucleotide-2 and origami from Particle-A, and exposingOligonucleotide-1, ii) exposed Oligonucleotide-1 hybridizes withOligonucleotide-3 to form Duplex 1-3, thereby releasingStored-Input-Oligonucleotide, and iii) step (i) reoccurs with theStored-Input-Oligonucleotide replacing the Input-Oligonucleotide untilthe reaction terminates. Typically the reaction terminates because theStored-Input-Oligonucleotide has all been released. By the reaction, theInput-Oligonucleotide is amplified by the release of theStored-Input-Oligonucleotide. Typically, the Input-Oligonucleotide isamplified by a factor of about 50 to about 1000.

The reaction can occur efficiently because the melting temperature (Tm)of Duplex Y-2 is greater than the Tm of Duplex 1-2; and the Tm of Duplex1-3 is greater than the Tm of Duplex 3-Y. For example, the Tm of DuplexY-2 is at least about 5% greater or at least about 20% greater than theTm of Duplex 1-2. The Tm of Duplex 1-3 is at least about 5% greater orat least about 20% greater than the Tm of Duplex 3-Y.

In another embodiment, a method for identifying anAnalyte-Oligonucleotide is provided. The method comprises providing aplurality of Particle-A and a plurality of Particle-B. Particle-A isfunctionalized with Oligonucleotide-1 and Oligonucleotide-2.Oligonucleotide-1 and Oligonucleotide-2 are hybridized to form Duplex1-2, and have attached oligonucleotide origami. The origami rendersParticle-A non-reactive. Oligonucleotide-1 is typically grafted ontoParticle-A. Particle-B is functionalized with Oligonucleotide-3 andTarget-Oligonucleotide. Typically, Oligonucleotide-3 is grafted ontoParticle-B. Oligonucleotide-3 and Target-Oligonucleotide are partiallyhybridized to form Duplex 3-Y. The Analyte-Oligonucleotide, Particle-Aand Particle-B are mixed, in any order. If the sequences of theAnalyte-Oligonucleotide and the Target-Oligonucleotide are substantiallyidentical (e.g. 99% or 100% identical), a cascade reaction occurs sothat: i) the Analyte-Oligonucleotide fully hybridizes withOligonucleotide-2 to form Duplex Y-2, thereby removing Oligonucleotide-2and origami from Particle-A, and exposing Oligonucleotide-1, ii) exposedOligonucleotide-1 partially hybridizes with Oligonucleotide-3 to formDuplex 1-3, thereby releasing Target-Oligonucleotide, and iii) step (i)reoccurs with the Target-Oligonucleotide replacing theAnalyte-Oligonucleotide. The Analyte-Oligonucleotide is identified asbeing the Target-Oligonucleotide if Particle-A and Particle-B aggregatein the reaction mixture.

In another embodiment, a sensor apparatus for identifying anAnalyte-Oligonucleotide is provided. The apparatus comprises a pluralityof Particle-A, and a plurality of Particle-B. Particle-A isfunctionalized with Oligonucleotide-1 and Oligonucleotide-2.Oligonucleotide-1 and Oligonucleotide-2 are hybridized to form Duplex1-2. Oligonucleotide Origami is attached to Duplex 1-2. The origamirenders Particle-A non-reactive. Oligonucleotide-1 is typically graftedonto Particle-A. Particle-B is functionalized with Oligonucleotide-3 andTarget-Oligonucleotide. Typically, Oligonucleotide-3 is grafted ontoParticle-B. Oligonucleotide-3 and Target-Oligonucleotide are hybridizedto form Duplex 3-Y. An Analyte-Oligonucleotide is mixed with Particle-Aand Particle-B, in any order. If at least a portion of the sequence ofAnalyte-Oligonucleotide is identical to the Target-Oligonucleotide, acascade reaction occurs so that: i) the Analyte-Oligonucleotide fullyhybridizes with Oligonucleotide-2 to form Duplex Y-2, thereby removingOligonucleotide-2 and origami from Particle-A, and exposingOligonucleotide-1, ii) exposed Oligonucleotide-1 partially hybridizeswith Oligonucleotide-3 to form Duplex 1-3, thereby releasingTarget-Oligonucleotide, and iii) step (i) reoccurs with theTarget-Oligonucleotide replacing the Analyte-Oligonucleotide. TheAnalyte-Oligonucleotide is identified as being theTarget-Oligonucleotide if there is an aggregation of Particle-A andParticle-B in the reaction mixture.

EXAMPLES Example 1

Begin with two 20 nm gold nanoparticles (AuNP), namely AuNP A (initialor deprotected particles A) and AuNP B (storage particles B), where eachmay be functionalized with ‘(red strand)’ first sequence strands and‘(purple strand)’ third sequence strands of DNA, respectively. Red(first sequence) strands may be grafted on AuNP A and purple (thirdsequence) strands may be grafted on AuNP B. There may be anapproximately 200 first sequence (red) strands on AuNP A andapproximately 200 third sequence (purple) strands on AuNP B. AuNP A, inaddition to the first sequence (red) strand, may be functionalized witha second sequence (green) strand. Part of the second sequence (greenstrand) of DNA may contain a sequence portion (5′ AAT CGC ATG C3′—underlined) which may be complementary to the part of first sequence(red) strand of DNA sequence

(5′ GCA TGC GAT T 3′-underlined).First sequence and second sequence (green and red) strands of AuNP A maybe partially hybridized. Due to selective hybridization between thefirst sequence and second sequence (red and green) strands particle(AuNP) A may bind to several origami structures as shown in FIG.1(b)(i). Particle (AuNP) A may be protected by the origami structures.

On the other hand, in FIG. 1(a), AuNP B functionalized with the thirdsequence (purple) strand, has a part of the third sequence ‘purplestrand’ that may contain a sequence (5′ GCT GCT GTA A 3′—underlined)which may be complementary to a part of a yellow (input) strand of DNAsequence (5′ TTA CAG CAG C 3′—underlined) of an input molecule. Theyellow (input) strand may have a specific inter strand hybridizationwith the third sequence (purple) strand. The specific inter strandhybridization may allow input molecule (yellow) to bind to storageparticle (AuNP) B as shown in FIG. 1(b)(ii).

DNA replacement reaction induced aggregation and isothermalamplification of DNA input molecule (yellow input strand) is shown inFIG. 1(b). First, in FIG. 1(b)(i), input DNA molecule (yellow strand)may fully hybridize with second sequence (green) strand (Tm ˜64.4° C.)of particle (AuNP) A (initial or protected particle). Partiallyhybridized second sequence and first sequence (green and red) strands(Tm ˜44.8° C.) may be displaced. This displacement may lead todeprotection of first sequence (red) strands grafted on now deprotectedparticle (AuNP) A (former initial or protected particles). The origamistructures bound to initial or protected particle (AuNP) A may bereleased. First sequence (red) strands of deprotected particle (AuNP) Amay be exposed.

The exposure of first sequence (red) strands of partially deprotectedparticle (AuNP) A may lead to a further DNA replacement inducedaggregation reaction as shown in FIG. 1(b)(ii). At this point, partiallycomplementary stored input molecule (yellow strand) and third sequence(purple) strands (Tm ˜42.3° C.) on storage particle (AuNP) B may bedisplaced by partially deprotected AuNP A and exposed first sequence(red) strands on partially deprotected particle (AuNP) A forming a firstsequence (red) and third sequence (purple) strand duplex (Tm ˜64.2° C.).Subsequent release of multiple output molecules (yellow strands) andaggregation of partially deprotected particles (AuNPs) A and storageparticles B may occur as shown in FIG. 1(b)(iii).

The formation of the duplex between third sequence (purple) strands andfirst sequence (red) strands may be energetically favorable and if thesetwo strands are able to hybridize then partially deprotected particles Aand storage particles B may bind (i.e., aggregate). Multiple binding ofparticles A and particles B may result in particle aggregation ratherthan isothermal amplification. Bare particles A and particles Bfunctionalized with first sequence (red) strands and third sequence(purple) strands respectively may aggregate upon mixing at roomtemperature as first sequence (red) and third sequence (purple) strandsmay hybridize with each other.

Input molecules (yellow (input) strands) of DNA may be added in anamount of 0.5 nM to the solution containing origami protected or initialparticles A and stored input molecules (yellow strands) hybridized withthird sequence (purple) strands grafted on particle B. The amount ofstored input molecules (yellow strands) may be 0.5 nM to 1000 nM. A 0.5nM or greater amount of yellow (input) strands may lead to a cascade ofreactions and may produce and release a larger number of outputmolecules of DNA (yellow strands) than input strands (yellow) asdelineated in FIG. 1(b).

As shown in FIG. 1(a), there may be a higher energy of hybridizationbetween input DNA molecules (yellow) strands and second sequence (green)strands on protected particles A as compared to second sequence (green)strands and first sequence (red) strands. Selective hybridizationbetween second sequence (green) strands and first sequence (red) strandson some protected particles A may be interrupted. One or more of the DNAorigami bound to protected particles A may be ejected from particles A.The removal of DNA origami from protected particles A or from thevicinity of protected particle A may lead to the activation of severalfirst sequence (red) strands on deprotected particle A which werepreviously dormant when under the protected particles A that had“umbrella” shielding of the DNA origami.

As shown in FIG. 1(b)(ii), the activated first sequence (red) strands onpartially deprotected particle A may hybridize with third sequence(purple) strands of storage particle B. Stored input molecules (yellowstrands) from storage particles B in the solution may be released. Asshown in FIG. 1(b)(iii), released output molecules (yellow strands) mayin turn release more DNA origami shields on remaining protectedparticles A, and that may promote a cascade of deprotection andhybridization events between partially deprotected particles A andstorage particles B. The outcome of the avalanche may be a liberation ofoutput molecules of single-stranded (ss) yellow output strands triggeredby a small amount of input molecules (yellow strands) thereby achievingan amplification of the input molecule (yellow strand). The cascadereaction may be irreversible.

Example 2—Fabrication and Characterization of AuNP-Origami Constructs(Protected Particles A)

AuNP-Origami constructs were fabricated by mixing 20 nm AuNPs A andrectangular origami together (several per particle) by mixing andletting origami hybridize on initial or deprotected particles A.

After purification by known methods such as for example gelelectrophoresis, atomic force microscopy (AFM) measurements were carriedout to characterize origami-AuNP conjugates (protected particles A). AnAFM image is shown in FIG. 2(b). The height profile extracted from AFMimaging shows several DNA origami structures with approximately 1.5 nmheight that are connected to an approximately 18 nm height sphericalAuNP (particles A) in high yield. There are very few unbound DNA origamifound in the area of view suggesting serial purification was successful.In FIG. 2(c) a histogram of number of origami bound to one particle A isshown and the distribution fits a Gaussian profile. On an average 3-4origami structures were found to be connected to one 20 nm AuNP A. Thehydrodynamic diameter of the construct was measured to be 200 nm withbroad size distribution using Dynamic Light Scattering (DLS) shown asblack trace in FIG. 3(d) which is significantly larger than that of onlyorigami (70 nm) and the AuNP A (30 nm). Amplification experiments wereconducted with these as prepared AuNPs with AuNP-origami constructs(protected particles A).

Amplification Experiments

An experiment was conducted in a test tube at room temperature by mixingparticles (AuNPs) A protected with origami (protected AuNP-A) withstorage particles B having stored input (yellow) strands on it(AuNP-B-Y). Particles A and B are in a 1:1 ratio, where Y representsthird sequence (purple) strands hybridized with stored input (yellow)strands on storage particle B. The concentration (amount) is 2.1 nM eachof particle A and particle B. The outcomes of the experiments wereverified by two methods, DLS measurements and steady state fluorescencespectroscopy. These inter-particle hybridization reactions werecompleted within one hour as shown in FIG. 4(c), as verified by in situfluorescence and DLS measurements.

DLS (Dynamic Light Scattering) Measurements.

The ensemble hydrodynamic diameter was measured of a 1:1 mixture withprotected particle A (protected AuNP-A) and storage particle B havingstored input (yellow) strands on it (AuNP-B-Y) [where Y denotes thirdsequence (purple) strand hybridized with stored (yellow) strands onstorage particle B where stored (yellow) strands have the same ordifferent DNA as yellow input strands]. The concentration is 2.1 nM foreach of protected particle A and storage particle B and is shown bytrace (b) in FIG. 3(d). The ensemble hydrodynamic diameter exhibitedsimilar size distribution for only protected AuNP A size distributionexcept there is a shoulder at lower size due to the presence of smallerAuNP-B-Y (hydrodynamic diameter approximately 30 nm). The ensemblehydrodynamic diameter did not change over time for up to several hoursvalidating a premise of steric protection of protected particle A fromreaction with storage particle B (i.e., protected particles A wereprotected against aggregation).

To the approximately 2 nM solution of each protected particle A andstorage particle B, different amounts of input molecules (yellowstrands) were added and kept at room temperature for 12 hours. Additionof 1 μM (1000 nM) input molecule (yellow strand) was found to lead toaggregation of aggregates of partially deprotected particles A withstorage particles B with sizes shown by trace (c3) in FIG. 3(d). Theamount of input molecule (yellow strand) was sequentially decreased byorders of magnitude and found that 100 nM (trace (c2)) and 10 nM (trace(c1)) concentration of input molecule (yellow strands) leads toaggregations of partially deprotected particles A with storage particlesB as shown by and trace respectively. The concentration of inputmolecule (yellow strand) decreased to 0.5 nM was found to lead tovisible aggregation of partially deprotected particles A with storageparticles B. However, the system without any input molecule (yellowstrand) added remained dispersed without signs of aggregations betweenprotected particles A with storage particles B. Thus, the DLSmeasurements showed that the presence of 0.5 nM amount of yellow inputstrand (which can be considered miniscule) leads to a cascade of stranddisplacement reactions, which, in turn, leads to aggregation ofpartially deprotected particles A and storage particles B, and therelease of output molecules (yellow strands) greater than inputmolecules (yellow strands,) i.e. amplification of input molecules(yellow strands). In order to gain a quantitative picture of thisamplification process, a fluorescence enhancement assay described in thefollowing section was developed.

Fluorescence Measurements:

In order to obtain a quantitative understanding of the number of outputmolecule (yellow DNA strands) released during the aggregation ofpartially deprotected particles A and storage particles B, Cy5 labeledyellow DNA was used. Cy5 has excitation maximum at approximately 650 nmand emission maximum at approximately 670 nm. The metal nanoparticlesurface is in close proximity to the bound fluorophores and typicallyexhibits a quenched fluorescence. However, when the fluorophores aredisplaced from the metal surface proximity, the fluorescence isrecovered back to the intrinsic fluorescence value. This method was usedto quantify the amount of output molecules (yellow DNA strands) releasedfrom the cascade reaction. The number of stored input molecules (yellowstrands) carried by storage particle B was estimated. Known 500 timesexcess of fluorophore modified yellow input strand was added to a knownconcentration in nanomolar range depending on particular analysis ofstorage particle B, for example, in the 1 to 10 nM range. Although 500times of Cy5-yellow DNA (Cy5) was added, it was found that approximately200 times remained in the solution as estimated by UV-Vis andfluorescence measurements of the supernatant. These results indicatedthat there are approximately 300 copies of stored input molecules(yellow DNA strands) on a single storage particle B. To figure out thequenching effect of 20 nm AuNPs on the Cy5, a strand displacement assaywas carried out. Different amounts of excess of first sequence (red)strands on partially deprotected particles A, which is fullycomplementary to the input molecules (yellow input strands) were addedto the AuNP-Y-Cy5 (labeled storage particle B) solution.

FIGS. 4(a) through (d) show results from a fluorescence enhancementassay for the determination of the number of DNA released. Due to highthermodynamic stabilization of duplex formation between first sequence(red) and third sequence (purple) strands, excess first sequence (red)strands can chase off input molecule (yellow strands) in the solutioncreating a separation between the fluorophore and AuNP shown in theupper panel of FIG. 4(a). The change in steady state fluorescence changeis shown in FIG. 4(a). In the presence of 300 times excess yellow inputstrands it is observed that the fluorescence signal of Cy5 getsamplified by approximately 1.85 times the initial intensity. In FIG.4(c), the relative fluorescence intensity change is plotted with theincreasing amount of the first sequence (red) DNA strand added. The plotshows steady increase of the fluorescence up to 200 times excess offirst sequence (red) DNA strands. As first sequence (red) DNA amountincreases no change of intensity was observed, and this suggests thatthe bound yellow-Cy5 DNA input strands were removed by the excess offirst sequence (red) DNA strands. The intercept of the black dotted linewith x-axis in FIG. 4(c), corresponds to the number of stored inputmolecule (yellow DNA) per particle. This observation further verifiesthat there are approximately 300 stored input molecules (yellow DNAinput strands) grafted on storage particle B.

Fluorescence enhancement assay was carried out to investigate how manyyellow-Cy5 strands were released during the aggregation of partiallydeprotected particles A and storage particles B in the absence orpresence of 0.5 nM input molecules (yellow DNA strands). During theaggregation process involving AuNPs A and B, all of the complementaryDNAs are not necessarily fully hybridized. Thus, a certain percentage ofoutput molecules (yellow strands) were released, as shown in the upperpanel FIG. 4(b). The amount of the output molecules (yellow DNA strands)released could be estimated from the fluorescence signal change in thesolution. The observed change was 1.2-1.4 times that of the initialintensity. The dotted lines in FIG. 4(c) indicate the number of releasedDNA, based on the fluorescence signal change. This result suggested that15-22% of the yellow-Cy5 input strands were released from the storageparticles B during the aggregation process. The amplification factor wascalculated as a number of output molecules (yellow strands) released(output) per one input molecule (yellow input strand) as 200-300, asshown in Table 1.

TABLE 1 Amplification factor is the ratio of amount of output strandreleased to input strand (or a number of output strands per one inputstrand) for the experimental realizations. Conc. of Conc. of Conc. of~Output DNA Particle A Particle B Input DNA Released Amplification (nM)(nM) (nM) (nM) Factor 2.1 2.1 0.5 300 * .15 * 2.1 191 2.1 2.1 0.5 300 *.22 * 2.1 277

Molecular Selectivity for Amplification Reaction:

The robustness of the present molecular amplification approach in thepresence of slightly different input molecules was investigated. Thisinvestigation tested the selectivity of amplification (i.e., onlytargeted or analyte input molecules are amplified) and how the systemcan withstand the interference from other non-targeted molecules. Thepoint mutations in DNA can produce very similar molecular species andmay not be distinguishable from the original DNA. These mutations mayrelate to the origin of genetic disorders or may be the cause forcancer. Robustness of amplification against sequence mutations maycontribute counterfeit detection, chemical safety, bio-sensing, DNAnanotechnology, information-computing and other applications. Improveddetection sensitivity of targeted molecules may be useful inbio-sensing, DNA nanotechnology, information-computing, counterfeitdetection, chemical safety and other applications.

The present amplification system was tested in the presence of themodified input molecules (yellow strands) with 1, 2 and 3 pointmutations. The point mutations are replacement of a single basenucleotide with another randomly chosen base (from the other threebases). Three sequences were selected to mimic the 1, 2 and 3 pointmutations shown in FIG. 5. At low concentrations of 10 nM, it wasobserved that the mutated input molecules (mutated yellow strands) arenot able to initiate the cascade reaction, as evident from the absenceof aggregations confirmed by no change in the DLS hydrodynamic diameter(FIG. 5). This experiment illustrates that the present amplificationmethod is capable of discriminating the signal from closely relatedsignals with near or at 100% specificity. Thus the present amplificationsystem can be utilized with other intricate amplification schemes andcircuits (analogues to electronics) that can perform complex operationswith an input molecular signal for a variety of applications.

Additional Embodiments

In one embodiment, a method is provided for isothermal molecularamplification comprising formulating a mixture of protected particles Acomprising DNA origami and of storage particles B, where protectedparticles A do not react with storage particles B, where storageparticles B are hybridized with stored input DNA molecule (yellow)having an input sequence, and where stored input DNA molecule (yellow)has the same input sequence as or has a portion of input sequence thatis the same as an input DNA molecule (yellow); adding the input molecule(yellow) to the mixture; releasing DNA origami from some of theprotected particles A and deprotecting some of the protected particlesA; iii. released origami reacting with input DNA molecule (yellow);deprotected particles A reacting with storage particles B and inducingrelease of stored input DNA molecule (yellow) from storage particle B asan output DNA molecule (yellow); and released output DNA molecule(yellow) mixing with some of the remaining protected particles Acreating a cascade reaction of ii. through iv. repeating until storedinput DNA molecules are released and deprotected particles A are fullydeprotected. In one embodiment, protected particles A are functionalizedwith first sequence (red) strands and second sequence (green) strands.In one embodiment, protected particles A are functionalized with firstsequence (red) strands and second sequence (green) strands that arecomplementary in sequence with one another. In one embodiment, partiallyhybridized first sequence (red) strands and second (green) strandspermit binding of origami to protect particles A. In one embodiment,protected particles A are functionalized with first sequence (red)strands and second sequence (green) strands and wherein input molecule Yfully hybridizes with second sequence (green) strands. In oneembodiment, partially hybridized first sequence (red) strands and secondsequence (green) strands permit binding of origami to protect particleA, wherein input molecule (yellow) fully hybridizes with second sequence(green) strands and displaces partially hybridized first sequence (red)strands and second sequence (green) strands, and wherein origami isreleased, protected particles A are deprotected and first sequence (red)strands are exposed. In one embodiment, protected particles A arefunctionalized with first sequence (red) strands and second sequence(green) strands, and storage particles B are functionalized with thirdsequence (purple) strands, wherein deprotecting particle A exposes firstsequence (red) strands, and wherein exposed first sequence (red) strandsform a duplex with third sequence (purple) strands. In one embodiment,protected particles A are functionalized with first sequence (red)strands and second sequence (green) strands, and storage particles B arefunctionalized with third sequence (purple) strands, whereindeprotecting protected particles A exposes first sequence (red) strands,and wherein exposed first sequence (red) strands form a duplex withthird sequence (purple) strands, and deprotected particles A bind withstorage particles B. In one embodiment, storage particles B arefunctionalized with third sequence (purple) strands. In one embodiment,storage particles B are functionalized with third sequence (purple)strands, and third sequence (purple) strands form a duplex with firstsequence (red) strands of deprotected particle A. In one embodiment,storage particles B are functionalized with third sequence (purple)strands and wherein third sequence (purple) strands are complementary insequence with part of stored input molecule (yellow). In one embodiment,partially hybridized third sequence (purple) strands of storageparticles B and stored input molecule Y permit binding of stored inputmolecule (yellow) to storage particle B. In one embodiment, theinvention is a DNA output molecule produced by the aforementionedmethods. In one embodiment, the present invention provides a sensorapparatus for detecting targeted molecules comprising one or moreprotected particles A, storage particles B and input DNA moleculeswherein protected particles A comprise first sequence (red) strands andsecond sequence (green) strands functionalized thereon, storageparticles B comprise third sequence (purple) strands grafted thereon andstored targeted DNA molecule (yellow) partially hybridized thereon,protected particles A, deprotected by input DNA molecules, react withstorage particles B by a duplex between third sequence (purple) strandsand first sequence (red strands), and input DNA molecules have amatching DNA sequence with stored targeted DNA (yellow) molecules andoutput DNA molecules (yellow).

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

INCORPORATION OF SEQUENCE LISTING

Incorporated herein by reference in its entirety is the Sequence Listingfor the application. The Sequence Listing is disclosed on acomputer-readable ASCII text file titled, “BSA 17-02 sequencelisting.txt”, created on Aug. 7, 2018. The BSA 17-02 sequence listingST25.txt file is 2 KB in size.

1. A method for amplifying an Input-Oligonucleotide, the methodcomprising a.) providing a plurality of Particle-A, wherein Particle-Ais functionalized with a plurality of Oligonucleotide-1 and a pluralityof Oligonucleotide-2, wherein Oligonucleotide 1 and Oligonucleotide 2are hybridized to form Duplex 1-2, and have attached oligonucleotideorigami; b.) providing a plurality of Particle-B, wherein Particle-B isfunctionalized with a plurality of Oligonucleotide-3 and a plurality ofStored-Input-Oligonucleotide, wherein Oligonucleotide-3 andStored-Input-Oligonucleotide are hybridized to form Duplex 3-Y, andwherein at least a portion of the sequences of the Input-Oligonucleotideand the Stored-Input-Oligonucleotide are identical; and c.) mixing theInput-Oligonucleotide, Particle-A and Particle-B, in any order, whereina cascade reaction occurs so that: i) the Input-Oligonucleotide fullyhybridizes with Oligonucleotide-2 to form Duplex Y-2, thereby removingOligonucleotide-2 and oligonucleotide origami from Particle-A, andexposing Oligonucleotide-1, ii) exposed Oligonucleotide-1 hybridizeswith Oligonucleotide-3 to form Duplex 1-3, thereby releasingStored-Input-Oligonucleotide, and iii) where (i) reoccurs with theStored-Input-Oligonucleotide replacing the Input-Oligonucleotide untilthe cascade reaction terminates, wherein the Input-Oligonucleotide isamplified by the release of the Stored-Input-Oligonucleotide.
 2. Themethod of claim 1, wherein the Tm of Duplex Y-2 is at least about 20%greater than the Tm of Duplex 1-2.
 3. The method of claim 1, wherein theTm of Duplex 1-3 is at least about 20% greater than the Tm of Duplex3-Y.
 4. The method of claim 1, wherein the cascade reaction terminateswhen all of the Stored-Input-Oligonucleotide is released.
 5. The methodof claim 1, wherein the Input-Oligonucleotide is DNA.
 6. The method ofclaim 1, wherein the Particle-A and the Particle-B are nanoparticlesfrom about 5 to about 100 nm in diameter.
 7. The method of claim 6,wherein the nanoparticles are selected from the group consisting of Au,Ag, Cu, Pt, Pd and combinations thereof.
 8. The method of claim 1,wherein the Input-Oligonucleotide consists of about 5 to about 120bases.
 9. The method of claim 1, wherein there are about 25 to about 400Oligonucleotide-1 attached per about 5 nm to about 40 nm diameter ofParticle-A and/or about 25 to about 400 Oligonucleotide-3 attached perabout 5 nm to about 40 nm diameter of Particle-B.
 10. The method ofclaim 1, wherein there are about 200 Oligonucleotide-1 attached perabout 20 nm diameter of Particle-A and/or about 200 Oligonucleotide-3attached per about 20 nm diameter of Particle-B.
 11. The method of claim1, wherein Oligonucleotide-1 is grafted to Particle-A, andOligonucleotide-3 is grafted to Particle-B.
 12. The method of claim 1,wherein the oligonucleotide origami is rectangular and wherein there areabout 2 to about 10 origami per about 5 nm to about 40 nm diameter ofParticle-A.
 13. The method of claim 12, wherein there are about 3 toabout 4 origami per about 20 nm diameter of Particle-A.
 14. The methodof claim 1, wherein the Input-Oligonucleotide is amplified by a factorof about 50 to about
 1000. 15. The method of claim 1, wherein thecascade reaction is under isothermal conditions.
 16. The method of claim1, wherein the relative amount of Particle-A:Particle-B is about 1:1.17. A method for identifying an Analyte-Oligonucleotide, the methodcomprising a.) providing a plurality of Particle-A, wherein Particle-Ais functionalized with a plurality of Oligonucleotide-1 and a pluralityof Oligonucleotide-2, wherein Oligonucleotide-1 and Oligonucleotide-2are hybridized to form Duplex 1-2, and have attached oligonucleotideorigami; b.) providing a plurality of Particle-B, wherein Particle-B isfunctionalized with a plurality of Oligonucleotide-3 and a plurality ofTarget-Oligonucleotide, wherein Oligonucleotide-3 andTarget-Oligonucleotide are hybridized to form Duplex 3-Y; and c.) mixingthe Analyte-Oligonucleotide, Particle-A and Particle-B, in any order,wherein if the sequence of the Analyte-Oligonucleotide and the sequenceof the Target-Oligonucleotide are identical, a cascade reaction occursso that: i) the Analyte-Oligonucleotide hybridizes withOligonucleotide-2 to form Duplex Y-2, thereby removing Oligonucleotide-2and oligonucleotide origami from Particle-A, and exposingOligonucleotide-1, ii) exposed Oligonucleotide-1 partially hybridizeswith Oligonucleotide-3 to form Duplex 1-3, thereby releasingTarget-Oligonucleotide, and iii) where (i) reoccurs with the TargetOligonucleotide replacing the Analyte-Oligonucleotide, wherein theAnalyte-Oligonucleotide is identified as the Target-Oligonucleotide byaggregation of Particle-A and Particle-B.
 18. The method of claim 17,wherein the Tm of Duplex Y-2 is at least about 20% greater than the Tmof Duplex 1-2.
 19. The method of claim 17, wherein the Tm of Duplex 1-3is at least about 20% greater than the Tm of Duplex 3-Y.
 20. A sensorapparatus for identifying an Analyte-Oligonucleotide, the apparatuscomprising a.) a plurality of Particle-A, wherein Particle-A isfunctionalized with a plurality of Oligonucleotide-1 and a plurality ofOligonucleotide-2, wherein Oligonucleotide-1 and Oligonucleotide-2 arehybridized to form Duplex 1-2, and have attached oligonucleotideorigami; and b.) a plurality of Particle-B, wherein Particle-B isfunctionalized with a plurality of Oligonucleotide-3 and a plurality ofTarget-Oligonucleotide, wherein Oligonucleotide-3 andTarget-Oligonucleotide are hybridized to form Duplex 3-Y, wherein theAnalyte-Oligonucleotide is mixed with Particle-A and Particle-B, in anyorder, wherein if at least a portion of the Analyte-Oligonucleotide isidentical to the Target-Oligonucleotide, a cascade reaction occurs sothat: i) the Analyte-Oligonucleotide fully hybridizes withOligonucleotide-2 to form Duplex Y-2, thereby removing Oligonucleotide-2and oligonucleotide origami from Particle-A, and exposingOligonucleotide-1, ii) exposed Oligonucleotide-1 partially hybridizeswith Oligonucleotide-3 to form Duplex 1-3, thereby releasingTarget-Oligonucleotide, and iii) where (i) reoccurs with theTarget-Oligonucleotide replacing the Analyte-Oligonucleotide, whereinthe Analyte-Oligonucleotide is identified as the Target-Oligonucleotideif there is an aggregation of Particle-A and Particle-B.